Radiation, Immune Checkpoint Blockade and the Abscopal Effect: A Critical Review on Timing, Dose and Fractionation

The combination of radiation and immunotherapy is currently an exciting avenue of pre-clinical and clinical investigation. The synergy between these two treatment modalities has the potential to expand the role of radiation from a purely local therapy, to a role in advanced and metastatic disease. Tumor regression outside of the irradiated field, known as the abscopal effect, is a recognized phenomenon mediated by lymphocytes and enhanced by checkpoint blockade. In this review, we summarize the known mechanistic data behind the immunostimulatory effects of radiation and how this is enhanced by immunotherapy. We also provide pre-clinical data supporting specific radiation timing and optimal dose/fractionation for induction of a robust anti-tumor immune response with or without checkpoint blockade. Importantly, these data are placed in a larger context of understanding T-cell exhaustion and the impact of immunotherapy on this phenotype. We also include relevant pre-clinical studies done in non-tumor systems. We discuss the published clinical trials and briefly summarize salient case reports evaluating the abscopal effect. Much of the data discussed here remains at the preliminary stage, and a number of interesting avenues of research remain under investigation

Osteoclast Activated FoxP3+ CD8+ T-Cells Suppress Bone Resorption in vitro

BACKGROUND: Osteoclasts are the body's sole bone resorbing cells. Cytokines produced by pro-inflammatory effector T-cells (T(EFF)) increase bone resorption by osteoclasts. Prolonged exposure to the T(EFF) produced cytokines leads to bone erosion diseases such as osteoporosis and rheumatoid arthritis. The crosstalk between T-cells and osteoclasts has been termed osteoimmunology. We have previously shown that under non-inflammatory conditions, murine osteoclasts can recruit naïve CD8 T-cells and activate these T-cells to induce CD25 and FoxP3 (Tc(REG)). The activation of CD8 T-cells by osteoclasts also induced the cytokines IL-2, IL-6, IL-10 and IFN-γ. Individually, these cytokines can activate or suppress osteoclast resorption. PRINCIPAL FINDINGS: To determine the net effect of Tc(REG) on osteoclast activity we used a number of in vitro assays. We found that Tc(REG) can potently and directly suppress bone resorption by osteoclasts. Tc(REG) could suppress osteoclast differentiation and resorption by mature osteoclasts, but did not affect their survival. Additionally, we showed that Tc(REG) suppress cytoskeletal reorganization in mature osteoclasts. Whereas induction of Tc(REG) by osteoclasts is antigen-dependent, suppression of osteoclasts by Tc(REG) does not require antigen or re-stimulation. We demonstrated that antibody blockade of IL-6, IL-10 or IFN-γ relieved suppression. The suppression did not require direct contact between the Tc(REG) and osteoclasts. SIGNIFICANCE: We have determined that osteoclast-induced Tc(REG) can suppress osteoclast activity, forming a negative feedback system. As the CD8 T-cells are activated in the absence of inflammatory signals, these observations suggest that this regulatory loop may play a role in regulating skeletal homeostasis. Our results provide the first documentation of suppression of osteoclast activity by CD8 regulatory T-cells and thus, extend the purview of osteoimmunology

Osteoclasts and CD8 T Cells Form a Negative Feedback Loop That Contributes to Homeostasis of Both the Skeletal and Immune Systems

There are a number of dynamic regulatory loops that maintain homeostasis of the immune and skeletal systems. In this review, we highlight a number of these regulatory interactions that contribute to maintaining homeostasis. In addition, we review data on a negative regulatory feedback loop between osteoclasts and CD8 T cells that contributes to homeostasis of both the skeletal and immune systems

The Abscopal Effect: A Review of Pre-Clinical and Clinical Advances

Radiotherapy has been used for more than a hundred years to cure or locally control tumors. Regression of tumors outside of the irradiated field was occasionally observed and is known as the abscopal effect. However, the occurrence of systemic anti-tumor effects was deemed too rare and unpredictable to be a therapeutic goal. Recent studies suggest that immunotherapy and radiation in combination may enhance the abscopal response. Increasing numbers of cases are being reported since the routine implementation of immune checkpoint inhibitors, showing that combined radiotherapy with immunotherapy has a synergistic effect on both local and distant (i.e., unirradiated) tumors. In this review, we summarize pre-clinical and clinical reports, with a specific focus on the mechanisms behind the immunostimulatory effects of radiation and how this is enhanced by immunotherapy

Tumor-draining lymph node is important for a robust abscopal effect stimulated by radiotherapy

Background Radiotherapy (RT) has been shown to stimulate an antitumor immune response in irradiated tumors as well as unirradiated distant sites (abscopal effect). Previous studies have demonstrated a role for the tumor-draining lymph node (LN) in mediating an anti-programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) stimulated antitumor immune response. Here, we investigated whether the LN is also important in mediating a RT alone stimulated abscopal response.Methods We used a subcutaneous modified B16F10 flank tumor model injected bilaterally. Our B16F10 cell line has an inserted viral glycoprotein which facilitated identification of tumor-specific T-cells. RT was directed at one flank tumor alone or one flank tumor and the tumor-draining LN. We evaluated response by tumor growth measurements and flow cytometry of both tumor-infiltrating and LN T-cells.Results We show that local tumor irradiation improves distant tumor control (abscopal effect). Depletion of CD8+ T-cells significantly reduced this abscopal response. We have previously shown, in a chronic lymphocytic choriomeningitis virus (LCMV) infection, that the T-cell proliferative burst following blockade of PD-1/L1 is provided by a ‘stem-like’ CD8+ T-cell subset which then differentiate into terminally differentiated effectors. These terminally differentiated effectors have the potential to kill virally infected or tumor cells following PD-1/L1 blockade. In the chronic LCMV infection, stem-like CD8+ T-cells were found exclusively in secondary lymphoid organs. Similarly, here we found these cells at high frequencies in the tumor-draining LN, but at low frequencies within the tumor. The effect of RT on this T-cell subset in unknown. Interestingly, tumor irradiation stimulated total CD8+ and stem-like CD8+ T-cell proliferation in the LN. When the LN and the tumor were then targeted with RT, the abscopal effect was reduced, and we found a concomitant reduction in the number of total tumor-specific CD8+ T-cells and stem-like CD8+ T-cells in both the irradiated and unirradiated tumor.Conclusions These correlative results suggest the tumor-draining LN may be an important mediator of the abscopal effect by serving as a stem-like CD8+ T-cell reservoir, a site for stem-like T-cell expansion, and a site from which they can populate the tumor

Exosomes, Their Biogenesis and Role in Inter-Cellular Communication, Tumor Microenvironment and Cancer Immunotherapy

Exosomes are extracellular vesicles ranging from 30 to 150 nm in diameter that contain molecular constituents of their host cells. They are released from different types of cells ranging from immune to tumor cells and play an important role in intercellular communication. Exosomes can be manipulated by altering their host cells and can be loaded with products of interest such as specific drugs, proteins, DNA and RNA species. Due to their small size and the unique composition of their lipid bilayer, exosomes are capable of reaching different cell types where they alter the pathophysiological conditions of the recipient cells. There is growing evidence that exosomes are used as vehicles that can modulate the immune system and play an important role in cancer progression. The cross communication between the tumors and the cells of the immune system has gained attention in various immunotherapeutic approaches for several cancer types. In this review, we discuss the exosome biogenesis, their role in inter-cellular communication, and their capacity to modulate the immune system as a part of future cancer immunotherapeutic approaches and their potential to serve as biomarkers of therapy response

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

Osteoclast-induced TcREG produce IL-10, IL-6, and IFN-γ.

<p>Mature osteoclasts (day 4) or osteoclast precursors were cultured with no Ag or OVA protein and then used to prime CD8 T-cells from an OT-I mouse. <b>A</b>. T-cells were collected at 48 h following initiation of co-culture. T-cells were stained for CD44, CD25 and FoxP3 and then analyzed by flow cytometry. T-cells co-cultured with osteoclasts in the absence of antigen do not express FoxP3⁺ or CD25 (top); FoxP3 and CD25 were induced in the presence of antigen as shown in the representative flow plot. The expression of FoxP3 was confirmed by reverse-transcription of RNA isolated from the co-culture and subsequent PCR of cDNA. GFP sorted cells from FoxP3^eGFP reporter mice were used as controls. Only mature (day 4) osteoclasts supported the generation of Tc_REG (right panel). <b>B</b>. Anti-mouse TGFβ was added to co-cultures at the dose indicated (left). Addition of recombinant murine TGFβ1 to co-cultures of CD8 T-cells and osteoclasts at concentration indicated (right). The percent of input T-cells converted to FoxP3⁺ are plotted in both panels. No statistically significant effect was observed on Tc_REG induction with either the addition of neutralizing antibody or recombinant TGFβ. <b>C</b>. Media was collected and cytokine quantitated by multiplexed ELISA. After 48 h of co-culture, cells were treated with Golgi stop and PMA plus ionomycin for 6 h. The cells were permeabilized, stained and evaluated for cytokine production by flow cytometry. While the CD11b⁺ osteoclasts were negative for all cytokines, the CD8⁺ T-cells stained triple positive for IL-10, IL-6, and IFN-γ. Statistical significance was assessed by non-parametric paired T test: *: P<0.05, **: P<0.01, ***: P<0.001 and ****: P<0.0001.</p

Tc_REG suppress mature osteoclasts by effecting the cytoskeletal reorganization:

<p>Osteoclasts were cultured either alone or with Tc_REG on bovine bone chips for 24 hrs. T-cells were then removed, and osteoclasts were stained with phalloidin-Texas Red to visualize actin rings. Representative images are shown in panel <b>A</b>. Quantitation of three independent experiments is shown in panel <b>B</b>. Panel <b>C</b> is quantitation of phalloidin staining of osteoclasts plated on tissue culture treated dishes. Statistical significance of actin ring area was assessed by non-parametric paired T test: **: P<0.01 in comparison to osteoclast alone.</p